正丁醇法制备大鼠脂肪组织细胞膜TSHR.pdfVIP

第13卷4期 天 津 医 科 大 学 学 报 Vo1．13．No．4 Dec．2007 485 2007年 12月 JOURNAL OF TIANJIN MEDICAL UNIVERSITY 正丁醇法制备大鼠脂肪组织细胞膜TSHR 马 彦，郭 刚，孙 蓓，辛 兢，赵学勤，张镜宇 (天津医科大学代谢病医院内分泌研究所，卫生部及天津市激素与发育重点实验室，天津300070) [摘要]目的：利用正丁醇对大鼠脂肪组织细胞膜上的促甲状腺素受体(TSHR)J~_行提纯，以获得膜受体的均一水溶性 溶液 方法：使用差速离心法获得大鼠含TSHR脂肪组织细胞膜碎片后，用正丁醇进一步提纯；采用SDS—PAGE和 ELISA法鉴定所得蛋白完整性、生物学活性，并用BCA法测定相关蛋白含量。结果：正丁醇提纯的大鼠TSHR结构完 整：经正丁醇提纯后的TSHR包被酶标孔孔间测定值变异系数较未经正丁醇提取组以及阳性对照组低。结论：经正丁 醇提纯后的大鼠TSHR可以满足临床ELISA法检测Graves病患者血清TRAb的要求，同时可以降低酶标孔间测定值 的误差。 f关键词]促甲状腺素受体；正丁醇；促甲状腺素受体抗体；酶联免疫吸附测定；大鼠 f中图分类号]R593 [文献标识码]A [文章编号]j∞6—8i47(2∞7)04—0485—04 Extraction of thyrotropin receptors from membrane preparations of the fat tissue of male rats with n-butanol MA Yan，GUO Gang，SUN Bei，XIN Jing，ZHAO Xue—qin，ZHANG Jing—yu (Institude of Endocrinology，Metabolic Disease Hospital，Tianjin Medical University；Key Lab of Hormones and Development，Ministry of Health and Tianjin，Tianjin 300070，China) ABSTRACT Objective：To develop a new method for extracting thyrotropin receptors(TSHR)from membrane preparations of the epididymal fat pads of male rats to get well—distributed water-solution． Methods：The crude Darticulate fraction containing rat fat cell membranes were prepared with differential centrifugati0n，then extracted with n—butano1．The integ“ty and bioactivity of TSHR from rat fat cell membrane were analyzed by means of SDS——PAGE and ELISA．Protein concentration was determined by using bicinchoninic acid solution(BCA)assays and BSA used as standard．Results：As desired，after extracted with n—butanol。TSHR didn’t lose it’S integrity．Well’S CV of extracted TSHR group was the lowest amang 3 groups (unextr